Evaluation of exotic soybean accessions and their use in developing improved soybean lines with resistance to Phomopsis seed decay

Poor seed quality of soybean is often associated with Phomopsis seed decay (PSD), which is one of the most economically important seed diseases. Diaporthe longicolla (syn. Phomopsis longicolla) is the primary cause of PSD. Control of PSD is best accomplished by planting PSD-resistant cultivars. Sixteen exotic soybean accessions from the USDA soybean germplasm collection were screened for reaction to PSD at Stoneville, Mississippi. They consisted of maturity groups (MG) II, III and IV. Seeds from inoculated and non-inoculated plots harvested either promptly at maturity, or after a two-week delay in harvest, were assessed for infection by D. longicolla. Seed infection ranged from 0 to 36.7%. Overall, PI 417050 (MG II), PI 417017 (MG III), and PI 594692 (MG IV) had significantly (P ≤ 0.05) lower percentages of seed infected by D. longicolla and higher seed germinations than other genotypes in the same maturity groups. PI 587982A also performed well. As a result of these findings, these resistant accessions were used over multiple cycles of breeding to develop improved breeding lines with resistance to PSD and low seed damage. Breeding line 11043-225-72, with combined resistance from both PIs 417050 and 587982A, had low scores for PSD (6.7%) and seed damage (3.4%), while DS65-1, deriving resistance from PI 587982A, had the lowest seed damage score (1.1%) and the highest seed germination (85.6%) among all lines tested in 2017. DS65-1 and 11043-225-72, along with five other improved breeding lines, were provided to public soybean breeders for developing improved cultivars and germplasm lines. DS31-243 (PI 700941), derived from PI 587982A, was publicly released by the USDA in 2022. This research will lead to future releases of improved germplasm lines and cultivars with PSD resistance and high seed quality. It will also aid in disease management and be a benefit to soybean producers and the industry at large.


Introduction
Soybean [Glycine max (L.) Merr.] is one of the most economically important legume crops grown world-wide [1]. It provides plant-based protein and oil for animal and human nutrition. utilizing host genetic resistance by planting resistant cultivars is one of the most effective methods for managing PSD in an environmentally friendly way [35][36][37][38][39]. Our long-term research goal is to develop high-yielding soybean cultivars with resistance to soybean diseases. To achieve this goal, the specific objectives of this study were (i) to evaluate unique exotic soybean accessions from the USDA germplasm collection for their reactions to D. longicolla infection after inoculation, followed by either prompt harvest at maturity or by a two-week delay in harvest after maturity; and (ii) to develop improved soybean breeding lines with PSD resistance and high seed quality.

Germplasm evaluation
Sixteen soybean plant introductions (PI) from the USDA Soybean Germplasm Collection were used in this study ( Table 1). Selection of these soybean lines was based on our previous screening of seed quality including, but not limited to, seed germination, frequency of hard seed and wrinkled seed, as well as the incidence of Phomopsis seed decay (PSD) in high-temperature environments with natural infection of PSD [40]. The selected soybean accessions consisted of maturity groups (MG) II, III, and IV, and included the susceptible checks PI 597412 [https:// npgsweb.ars-grin.gov/gringlobal/methodaccession?id1=51091&id2=494138] for MG II, PI 417077 for MG III trials [40], and PI 80837 for MG IV [41]. The resistant checks included PI 417274 [42], PI 417017 [43], and PI 417479 [39] for the trials of MG II, III, and IV, respectively.
Field experiments were conducted at Stoneville, Mississippi (MS) on a Sharkey clay soil (very-fine, smectitic, thermictic Chromic Epiaquert) from 2006 to 2007. For the germplasm evaluation experiments, single-row plots were planted on 18 April 2006 and 20 April 2007. The seeding rate was 33 seeds/m of row in 2.74 m-long rows, with a 0.91-m row spacing. Each plot was a single genotype with a specific inoculation/harvest date treatment. Maturity groups II, II, and IV were utilized and evaluated as three separate experiments. Within each of these experiments, there was one inoculated block and one non-inoculated block. Each experiment of maturity group and inoculation was a split-plot arrangement of treatments in a randomized complete block design (RCBD), with soybean genotypes as main plots, being randomly arranged within each of six replications, and harvest time (prompt versus two-week delay) as sub-plots.
Each plot was manually bulk-harvested at the appropriate time. Specifically, in 2006, the following treatment combinations were carried out: prompt harvest-inoculated, prompt harvestnon-inoculated, and delayed harvest-inoculated. In 2007, the same treatment combinations were employed with the added combination of delayed harvest-non-inoculated. No chemical treatments were applied to the experiments, as their application to plots inoculated with a soybean pathogen could bias the PSD results. Hence, weed control was manual.
At the R5 growth stage [44], inoculum of D. longicolla, as described below, was applied twice each year at an interval of 14 days. To promote pathogen infection, water was applied as needed by either manually spraying with a battery-operated Solo Model 416 backpack sprayer (Flojet Co., Santa Ana, CA) or through furrow irrigation.
Weather data of total precipitation, number of rainy days, average maximum temperatures and maximum relative humidity during the soybean growing seasons were obtained from the Stoneville, MS weather station [http://deltaweather.extension.msstate.edu/ag-weather-tools].

Breeding populations
As part of an ongoing effort to incorporate the highest levels of resistance from diverse exotic accessions into improved breeding lines, over 100 heterogeneous breeding lines were evaluated annually over multiple years. Crosses were made between high-yielding soybean lines and the newly identified exotic sources of PSD-resistance, as well as between the new sources of resistance to PSD and previously developed heat-tolerant soybeans [45,46]. Although hundreds of breeding lines were developed and tested during the course of the project [47], this work focuses on the results of the following seven improved breeding lines: 11043-225-72, 11043-224-91, 11030-541-28, 10061-236-11, 10076-121-11, DS65-1, and DS31-243. Each of these lines was derived from resistant accessions tested above (PI 587982A and PI 417050) as well as from an additional resistant accession, PI 424324B, that was identified previously by Li et al. [9]. Attempts were also made to create improved lines derived from multiple sources of resistance, such as PI 587982A with PI 424324B and PI 587982A with PI 417050. Susceptible lines were dropped when identified, while the best of these lines went on for additional testing in yield trials and were later shared with public soybean breeders. Lines 11043-225-72 and 11043-224-91 were derived from DS25-1 x PI 417050. DS25-1 [45,46] is an improved heat tolerant soybean germplasm line released by the USDA in 2017 [https://npgsweb.ars-grin.gov/ gringlobal/accessiondetail?id=1954416]. It was derived from DT98-9102 x PI 587982A, where PI 587982A was used in the cross because of its heat tolerance, high germinability, and potential resistance to PSD [40]. DT98-9102 is an improved high-yielding germplasm line developed by R. L. Paris at Stoneville, MS and released by USDA, but not entered into the USDA germplasm collection. PI 417050 had high germinability and low PSD in a previous study [40]. Breeding line 11030-541-28 was derived from DS25-1 x PI 424324B, where PI 424324B was previously identified to be resistant to PSD [9]. Line 10061-236-11 was derived from breeding line 04030-1-4-5-1 x LG03-4561-14 [48]. Breeding line 04030-1-4-1-1 [49] is heat tolerant and was derived from cultivar 5601T [50] x PI 587982A. Breeding line 10076-121-11 was derived from line 07055-2-3-7 x 'Osage' [51], where breeding line 07055-2-3-7 was derived from one backcross with DT97-4290 [52] x PI 587982A. Breeding line DS65-1 was derived from PI 587982A x S99-11509 [53] and breeding line DS31-243 was derived from LG04-1459 x 07055-2-3-7. LG04-1459 is a high-yielding, exotically-derived unreleased breeding line developed by R. L. Nelson (USDA, retired) at Urbana, IL. All crosses were made at Stoneville, MS during summer seasons and the F 1 plants were grown in Puerto Rico during the following winter/ spring of the year. Plants were advanced from the F 2 to F 3 , F 3 to F 4 , F 4 to F 5 , F 5 to F 6 and any succeeding generations by pedigree selection [54] at Stoneville, MS. In each generation, selection of individual plants was based on the best agronomic traits (lodging, height, lack of shattering, pod load, pod color, etc.) in the field, followed by assays for germination and/or PSD in the lab. Only plant progenies with the best agronomic traits, seed germination, and low PSD scores were advanced to the next generation.
In single-replicated field trials in 2015 and 2016, each plot was a single row 2.7 m long and 0.91 m wide. All plots were bordered with other experimental lines or with 'Williams 82.' Twenty-five seed m -1 of row were planted at a depth of 2.5 cm using a tractor-drawn planter fitted with seed cone attachments and a hydraulic depth-control system. Plots were furrow irrigated as needed every 10-14 days throughout the growing season, which created humidity that rose from the soil through the plant canopy.
Plants of breeding lines were inoculated twice during the period from R5 through R6 [44]. Single plants were selected from heterogeneous F 3 , F 4 , F 5 and later-generation rows with the best agronomic traits shortly after R8. Homogeneous rows at the F 5 and later generations were harvested in bulk. Harvested plant materials were placed in an air-conditioned humidity-controlled building, where they were stored and allowed to uniformly dry, preparatory to being threshed in a single-plant or bundle thresher as appropriate. Once threshed, seed was stored at 21˚C and 60% relative humidity until they were assayed for PSD infection and/or seed germination.
In 2017, advanced breeding lines 11043-225-72, 11043-224-91, 11030-541-28, 10061-236-11, 10076-121-11, and DS65-1, and commercial cultivars Pioneer 94Y23, AG4403, AG4232, and Progeny 4211, were grown in replicated four-row plots at Stoneville, MS. Planting date was 10 April and row length was 5.79 m long, but the other planting protocols were as described above. The experimental design was a randomized complete block design with three replications. Due to excessive rains and warm temperatures during August and September in 2017, plots were not inoculated or delay-harvested. The middle two rows of each plot were promptly harvested shortly after R8. Harvested seed was then stored and assayed as per the protocols below. Seed were assayed for PSD, standard germination, and damage kernel total (DKT), as defined by the Federal Grain Inspection Service [https://www.ams.usda.gov/aboutams/programs-offices/federal-grain-inspection-service]. To determine total seed damage (DKT), seed were visually rated for mold, stink bug feeding, green seed, weathering, and heat damage on seed lots of 125 g. Percent seed damage represents the total weight of damaged seed divided by 125 g x 100.

Inoculum preparation and application
Isolate LiMS-1 of D. longicolla, which was available in 2006, was used for inoculation in the germplasm evaluation experiments, whereas another isolate MSPL 10-6 was used for the breeding trials. MSPL 10-6 was one of the most aggressive isolates in our tests [17]. Both isolates were originally from field-grown soybean plants at Stoneville, MS. To prepare the inoculum, the fungal isolate was grown at 24˚C on potato dextrose agar (Difco Laboratories, Detroit, MI). After autoclaving, the medium was adjusted to pH 4.8 with 25% lactic acid (APDA). Inoculum was prepared using our previously reported method [9,10]. Briefly, sporulation of the culture was induced under a fluorescent light output of 300 μmol m -2 s -1 with a 12-h photoperiod for 30 to 45 days. Sporulating cultures in Petri dishes were then flooded with sterile deionized water three times, agitated to dislodge conidiospores and filtered with four layers of sterile cheesecloth to eliminate the agar. Concentrations of conidiospores were adjusted to approximately 1.5 x 10 5 /ml, which was determined using a hemacytometer (Hausser Scientific, Blue Bell, PA). A battery-operated backpack sprayer (Solo Model 416; Flojet Co., Santa Ana, CA) was used for field inoculations. The sprayer had a hand-held boom containing a single nozzle with an adjustable orifice at 207 kPa. In each plot, a conidiospore suspension was sprayed directly onto the soybean pods and then evenly across the foliage. Plots were sprayed until runoff [9,10,55]. Approximately 500 ml of the conidiospore suspension was applied to each plot.

Seed assays
Prior to planting, 25 seeds of each line were plated and assayed for incidence of D. longicolla. After manual harvest and threshing of plants, 25 randomly chosen seeds (13% moisture) from each plot (i.e. each replication) for each soybean line for each inoculation treatment/harvest time combination were assayed to determine the percent seed infection by D. longicolla, using the method previously reported [9,10,55]. Seeds were surface-disinfected in 0.5% sodium hypochlorite for 3 min, rinsed in sterile distilled water, and then placed on APDA [9,10,55]. Five seeds were placed on APDA in each 100 mm x 15 mm Petri dish, where one seed was placed in the center and the others were placed equidistantly around the outside of the dish, approximately 10 mm from the side with approximately 30 mm between seeds. All seed plates were incubated for four days at 24˚C. The number of seed infected with D. longicolla was recorded and calculated as percent seed infection. Seed germination of 200 arbitrarily selected seeds from each line was determined using a standard soybean seed germination protocol [56].

Isolate identification
Putative D. longicolla colonies were transferred to new APDA plates. In order to distinguish D. longicolla from other Diaporthe spp. in the Diaporthe-Phomopsis complex, such as D. sojae (syn. Phomopsis phaseoli), Diaporthe caulivora and Diaporthe aspalathi (syn. Diaporthe phaseolorum var. meridionalis), thirty selected seed plates with putative D. longicolla colonies were maintained for 45 days at 24˚C under 12-h light. In addition, 10 putative D. longicolla isolates, along with the type strain TWH P74, LiMS-1, and isolate MS-SSC91 of D. aspalathi (syn. Diaporthe phaseolorum var. meridionalis) [57] were transferred to PDA or water agar with autoclaved soybean stem pieces or Williams 82 seeds for 45 days under incubation conditions. Observations under the Olympus SZX12 dissecting microscope were conducted beginning 10 days after incubation to check if any isolates formed perithecia. Perithecia have not been found in D. longicolla, but have been reported to be produced by other fungal pathogens in the Diaporthe-Phomopsis complex, such as D. aspalathi that causes southern soybean stem canker, and D. caulivora (syn. Diaporthe phaseolorum var. caulivora) that causes northern soybean stem canker [58], as well as D. sojae (syn. Phomopsis phaseoli) that causes soybean pod and stem blight [59].

Data analyses
Quantitative data were analyzed statistically and analyses of variance were calculated using the Generalized Linear Mixed procedure (PROC GLMMIX) in SAS (version 9.4, SAS Institute, Cary, NC) with a negative binomial distribution and a log link function specified for seed infection by D. longicolla [62]. Genotype, inoculation treatment, harvest date, and year were independent variables, whereas incidence of D. longicolla, percent seed germination, and percent damaged seed (DKT) were dependent variables. Mean percent seed germination was calculated for each entry. Data were combined across years to test if there were interactions between genotype and year when "year" was the fixed effect, and to compare genotypes within a year, in which "year" was the random effect and "genotype" was the fixed effect. The genotypes were compared with Fisher's least significant difference (LSD) at P � 0.05 to determine differences among them. Pearson's correlation coefficients between percent seed infected by D. longicolla and percentage of seed germination were computed using the PROC CORR procedure of SAS. .7˚C, respectively, which were much higher than 24˚C, the optimal temperature for PSD. In 2006, totals for precipitation in June, July, and August were 46, 45, and 40 mm, respectively, while in 2007, totals for precipitation in those months were 99, 197, and 87 mm, respectively (Fig 1).

Evaluation of exotic accessions for incidence of D. longicolla and percent seed germination
Results of seed assays prior to planting indicated that the soybean accessions we selected had good seed quality. They were free of D. longicolla. However, the PSD-causing pathogen was found in seeds harvested from the field inoculation experiments. Putative isolates of D. longicolla that had typical morphology similar to those of the type strain TWH P74 (ATCC 60325) and isolate LiMS-1 were recorded for each seed tested and used to determine the percentage of seed infection by D. longicolla. Observations of 45-day-old cultures of 30 putative isolates of D. longicolla under the Olympus SZX12 dissecting microscope, on either PDA plates from the seed plating assay or water agar with soybean stem pieces, indicated that no perithecia were present. However, the stem canker-causing pathogen D. aspalathi, isolate MS-SSC91, did form perithecia in the side-by-side comparison with isolates of D. longicolla. Other unidentified Diaporthe/Phomopsis spp., as reported by Mengistu et al. [12], as well as Fusarium spp. and Alternaria spp., were found infrequently. Because soybean plants were inoculated with D. longicolla in our field experiments, and D. longicolla is the prevalent species, the data that we present from this study are focused only on this species.
DNA sequences of four selected isolates were identical with our previous D. longicolla isolates, deposited to Genbank, at the ITS region (Accession MF134860) and in the TEF-1α gene (Accession MF189565) [55]. They also have 99-100% identity with many other reported sequences of D. longicolla strains in GenBank, which included, but were not limited to, accession NR_144924 for the type strain TWH P74 at the ITS regions, and Accessions 590766 and HQ445912 for isolates FAU657 and ER 1678 in the TEF-1α gene, respectively. It is notable that when the sequences of our D. longicolla isolates were compared with D. sojae isolates, such as FAU499 (Accession KJ590760) and FAU604 (Accession KJ590759), there was only 84% similarity of sequence between those two fungal pathogens.
Analysis of variance of seed infection by D. longicolla for MG II, MG III and MG IV accessions indicated that there were significant differences (P � 0.05) among accessions, harvest times, and years. There were significant (P � 0.05) interactions of accession × year, and accession × harvest × year. In MG III and MG IV, interactions of treatment × year were found. For seed germination, there were significant differences (P � 0.05) among accessions, harvest  80837 (MG IV) were 0, 0, and 3.3%, respectively, while in the prompt-harvest inoculated treatments, their corresponding percentages of seed infection by D. longicolla were 5.8, 16.7, and 36.7%, respectively (Table 2). However, PI 417328 had similar percentages of seed infection by D. longicolla in both the delay-harvest non-inoculated and delay-harvest inoculated treatments, with 12.0% and 12.7%, respectively in 2007 (Table 2). There was a significant (P � 0.05) time-of-harvest effect. In general, D. longicolla seed infection was higher in plots with delayed harvest than in those with prompt harvest. For example, a susceptible accession PI 597412 (MG II) had 11.7% seed infection when harvested on time and 22.5% infection when delay harvested from the inoculated plots in 2006. Percentages of D. longicolla seed infection of PI 417017 (MG III) of inoculated plots in 2007 were 3.3% and 14.0% in prompt and delayed-harvest tests, respectively. In the non-inoculated plots, PI 587576 (MG IV) had seed infection of 5.3% and 11.3% in prompt and delayed-harvest tests, respectively (Table 2). However, there were exceptions. For example, PI 594692 had the same 2.5% of seed infection by D. longicolla in both harvest schemes in non-inoculated plots in 2006.
In view of the results of inoculation treatments and harvest times across both years, PI 417050 (MG II), PI 417017 (MG III), and PI 594692 (MG IV) had significantly (P � 0.05) lower percent seed infected by D. longicolla than most other soybean genotypes in the same maturity group test (Table 2). In addition, soybean genotypes PI 416875, PI 417321, PI 423941 and PI 417274 in MG II, PI 85009-1, PI 417328, and PI 587982A in MG III, and PI 587576, PI 594872 and PI 417479 in MG IV had significantly (P � 0.05) lower percent seed infected by D. longicolla than their respective susceptible checks in their respective maturity group tests ( Table 2). There were differences in seed germination among accessions (Table 3). In general, inoculation with D. longicolla decreased seed germination. In 2006, percent seed germination of PI 417274 (MG II) in the non-inoculated treatment with prompt harvest was 98.3%, while it was 86.7% in the inoculated test (Table 3). In 2007, with prompt harvest, PI 417017 (MG III) and PI 594692 (MG IV) were other good examples. They had 96.0 and 85.7% seed gemination in the non-inoculated treatment, respectively. However, their percentages of seed germination in the inoculated treatment were both reduced to 77.3% and 57.7%, respectively. In some cases, inoculation treatments did not cause significant differences, such as for the susceptible check PI 417077 in the MG III prompt-harvest trial in 2006, where the percentages of seed germination in both non-inoculated and inoculated tests were 45% (Table 3). Harvest times could also affect seed germination. It appeared that delayed harvest decreased seed germination in most cases. In MG II trials in 2007, PI 417050 had 86.0% and 83.0% seed germination in non-inoculated and inoculated treatments, respectively, when harvested promptly, but 78.4% (non-inoculated) and 76.0% (inoculated) seed germination in the delayed harvests (Table 3).
Results from correlation analyses indicated that percent seed infected by D. longicolla was significantly (P < 0.05) and negatively correlated with seed germination in almost every inoculation treatment and harvest time across both 2006 and 2007 (Fig 2).

Evaluation of improved breeding lines
Lines 11043-225-72, 11043-224-91, and 11030-541-28 were tested in 2015 and 2016 for PSD and seed germination. Although the scores were all zero for PSD in 2015 and were low, ranging from 8.0 to 28.0%, in 2016, there were not adequate checks for comparison (Table 4). However, for seed germination, there were multiple appropriate susceptible checks (CZ3841LL, LG03-4561-14, and LD06-7620), with germination scores of 51.5%, 46.0%, and 34.0%, respectively in 2016, and similar scores in 2015, although lacking data for CZ3841LL for that year (Table 4). In comparison, the seven improved breeding lines all had germination percentages in the 90s in both 2015 and 2016, indicating that they all had high germinability and good seed quality in stressful environments.

Discussion
Seed quality is very important for soybean growers and processors. Poor seed quality of soybean is often associated with Phomopsis seed decay (PSD). In this study, experiments were designed to evaluate 16 maturity group II, III and IV exotic soybean accessions as a first step toward developing improved soybean germplasm lines. The soybean accessions selected for this study showed different levels of natural field infection from D. longicolla in high-  [40]. We therefore hypothesized that PSD-resistant gene(s) may exist in these selected soybean lines. Furthermore, we surmised the new sources of resistance to PSD could be identified under controlled inoculation treatments and utilized for breeding improved germplasm lines with resistance to PSD. Field trials without inoculation may risk plants escaping pathogen infection if the pathogen is not evenly distributed in the field, which would lead to false conclusions in identifying resistant sources [10,55]. Artificial inoculation under controlled conditions can provide a more uniform distribution of the pathogen and reduce the incidence of disease "escapes" when evaluating a genotype's reaction to the pathogen. It also provides disease pressure for confirming disease resistance of soybean genotypes [9,64,65]. In this study, soybean genotypes were evaluated under inoculated and non-inoculated treatments. Significant differences in percentages of seed infection by D. longicolla enabled identification of resistant genotypes to PSD in all three maturity groups of soybeans. PI 417050 (MG II), PI 417017 (MG III), and PI 594692 (MG IV) had significantly (P � 0.05) lower percentages of seed infected by D. longicolla than their respective susceptible checks, as well as compared to other genotypes in the same maturity group tests. There were no specific effects of seed infection, germination, or damage due to maturity group in this study, as maturity and genotype were confounded (each genotype has only one maturity).
Populations with resistant accessions were made in 2006 and tested in subsequent generations. Although not as numerically low in PSD as the above three genotypes, other accessions, such as PI 587982A, also had significantly lower PSD values than the susceptible checks. PI 587982A was used extensively in breeding for PSD and heat resistance and is therefore in the parentage of each of the lines in Table 4. Additionally, PI 417050, in combination with PI 587982A, was used to develop breeding line 11043-225-72, which had among the lowest observed levels of both PSD (6.7%) and DKT (3.4%) among all lines tested in 2017 (Table 4). Further, DS65-1 had the highest germination (85.6%) and the lowest level of DKT (1.1%) in 2017, and was the only line tested that had seed damage levels less than the typical price-   PLOS ONE discount level of 2.1%. These sources of resistance to PSD are highly useful in soybean breeding programs and have been extensively utilized. Colonization of D. longicolla on soybean and development of PSD are very sensitive to environmental conditions and prefer warm and humid conditions [23,26,66]. In this study, the incidence of PSD was higher in 2007 than in 2006, likely because of environmental factors. When examining weather data, average maximum air temperatures during the soybean growing seasons of 2006 and 2007 were similar, but there were large differences in total precipitation. In July, when most soybean plants reached the R5 growth stage and seeds begin to develop in pod cavities [44], the total precipitation in 2006 was 45 mm, while in 2007, it was 196 mm. During the harvest season in August, the total precipitation in 2006 was 45 mm, while in 2007, it was 87 mm. The difference in late season rainfall between 2006 and 2007 was likely the main cause for differences in PSD incidence between those two years.
The "early soybean production system" (ESPS) is very popular in the midsouthern United States [67]. In this system, early maturing cultivars are planted in late-March through April, and mature in August through September. The ESPS allows soybean to take advantage of plentiful spring and early summer rains to increase chances of avoiding late-season drought, thereby increasing soybean yield [67]. However, the weather in August is usually hot and humid, which can result in severe PSD and resulting poor seed germination in the south [68]. Rainfall or moisture after maturity has been reported to be the key factor determining the extent of damage by D. longicolla and other seedborne pathogens [69]. Delayed harvest due to rainfall with high humidity usually leads to higher infection levels [70]. In this study, the effect of harvest timeliness (prompt vs. delayed) on the incidence of PSD was analyzed. Our results showed that mean seed infection by D. longicolla was more severe after delayed harvest compared to prompt harvest at maturity. Additionally, genotypes that had low seed infection by D. longicolla when harvested on time, could show susceptibility to D. longicolla when harvest was delayed. For example, PI 594872 (MG IV) in 2007 had 2.7 and 8.0% seed infection by D. longicolla in the non-inoculated and inoculated prompt-harvest treatments, respectively. However, when harvest was delayed, the percentages of seed infection by D. longicolla for this accession were as high as 8.7% and 20.7% for the non-inoculated and inoculated treatments, respectively. Therefore, evaluating soybean genotypes under a delayed harvest regime or under conditions that favor PSD disease development is the preferred approach to identify PSD-resistant soybean lines.
Seed gemination is one of the important characteristics of seed quality. In many cases, the percent seed infection by D. longicolla is a good predictor of seed gemination. The most resistant genotypes, PI 417050 (MG II), PI 417017 (MG III), and PI 594692 (MG IV) not only had significantly (P � 0.05) lower percent seed infected by D. longicolla, but also had higher seed germination than the other genotypes in the same maturity group. All susceptible soybean checks in the three maturity groups tested had poor germination, especially in inoculated trials with delayed harvest, which is consistent with our previous findings [9,10,29,55]. Poor seed germination of most soybean cultivars in the ESPS is typical because the current cultivar gene pool apparently lacks adequate resistance genes to PSD and heat [40]. However, identifying resistance to PSD and heat in exotic accessions, and then using those accessions to breed resistance into improved cultivars, appears to be a promising approach.
Developing and utilizing PSD-resistant cultivars is an economical and environmentally friendly strategy to protect soybean crops from disease-induced seed damage, especially when using the popular ESPS in midsouthern states. Due to an extended and committed effort, twenty-seven PSD-resistant homogeneous breeding lines with resistance to PSD and high seed quality were identified from among hundreds of breeding lines, and then tested in multi-year trials. Among these, seven soybean lines, 11043-225-72, 11043-224-91, 11030-541-28, 10061-236-11, 10076-121-11, DS65-1, and DS31-243, were transferred to public soybean breeding programs for developing improved cultivars and germplasm lines. DS31-243 (PI 700941) was publicly released by the USDA in 2022. It is expected that this research will lead to future releases of improved germplasm lines and cultivars with PSD resistance and high seed quality through the use of one or more of the above seven lines, which are all available from the ARS authors to public and commercial researchers through material transfer agreements. In terms of recommendations, DS65-1 and DS31-243 represent the best of these lines derived solely from PI 587982A, whereas 11043-225-72 is the best line derived from PI 417050 and PI 587982A, and 11030-541-28 is the best line derived from PI 424324B and PI 587982A.
D. longicolla has been identified as the main causal agent of PSD. However, although it was the dominant species in isolates from soybean in multiple studies [11][12][13], several different fungi in the Diaporthe-Phomopsis complex have also been found to be associated with PSD in soybean [11,71,72]. Evaluating soybean breeding lines with a panel of different fungi from the Diaporthe/Phomopsis complex would aid in the management of soybean seed decay, breeding for resistance, and be a benefit to soybean producers and the industry at large.